Publicação
Engineering a Galactose Oxidase for Enlarged Biotechnological Applications
| Resumo: | Lignocellulosic biomass (LCB) is a promising source of feedstocks that can substitute fossil petroleum-based fuels and chemicals, since it is renewable, abundant and their utilisation does not compete with food crops. From the fractionation of LCB, lignin, cellulose and hemicellulose monomers can be bio-transformed into final products that are of biotechnological interest. Galactose oxidase (GalOx), harbouring a copper radical cofactor, regioselectively catalyse the oxidation of D-galactose, but also of primary alcohols (like benzyl alcohol) and furans (like hydroxymethylfurfural, HMF) prevenient from LCB, using O2 as the co-substrate. In this work, we aimed to improve the activity of PsGalOx, a bacterial enzyme recently identified in the lab, towards the oxidation of interesting chemical precursors such as benzyl alcohol. Directed evolution steps were optimised and after thousands of variants screened, two variants (14G3 and 41C7) were found in the two sequential rounds performed. Variant 14G3 carries only mutation N511S and variant 41C7 carries additionally the mutation D446G. The purified enzyme preparations demonstrated that 14G3 exhibited a significant 10- to 20-fold increase in catalytic efficiency (kcat/Km) across the three substrates (benzyl alcohol, D-galactose and HMF), when compared to its parent. This enhancement is likely due to alterations in the active site architecture, as the N511S mutation is positioned in close proximity to this region. Nevertheless, the mutation introduced in 41C7 did not affect the catalytic performance, yet a 2-fold increase in protein production was observed. Both variants showed a slight loss in thermostability, suggesting a trade-off between activity and stability. Preliminary tests on immobilized enzymes in metal affinity resins showed that purified enzymes could be successfully attached to the carrier, however, a drop of activity was observed upon immobilisation. The nickel resin showed better results than cobalt resin, maintaining its activity even after 30 days of storage at 4 ºC. Further optimisation and development of enzyme immobilisation need to be performed to increase the efficiency of the enzyme when immobilised and, eventually, the stability to make it applicable for larger-scale applications in biotechnological industries and biorefineries. |
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| Autores principais: | Jesus, Pedro Miguel Brito de |
| Assunto: | lignocellulosic biomass carbohydrate-active enzymes galactose oxidase directed evolution catalytic efficiency sustainable chemistry |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Lignocellulosic biomass (LCB) is a promising source of feedstocks that can substitute fossil petroleum-based fuels and chemicals, since it is renewable, abundant and their utilisation does not compete with food crops. From the fractionation of LCB, lignin, cellulose and hemicellulose monomers can be bio-transformed into final products that are of biotechnological interest. Galactose oxidase (GalOx), harbouring a copper radical cofactor, regioselectively catalyse the oxidation of D-galactose, but also of primary alcohols (like benzyl alcohol) and furans (like hydroxymethylfurfural, HMF) prevenient from LCB, using O2 as the co-substrate. In this work, we aimed to improve the activity of PsGalOx, a bacterial enzyme recently identified in the lab, towards the oxidation of interesting chemical precursors such as benzyl alcohol. Directed evolution steps were optimised and after thousands of variants screened, two variants (14G3 and 41C7) were found in the two sequential rounds performed. Variant 14G3 carries only mutation N511S and variant 41C7 carries additionally the mutation D446G. The purified enzyme preparations demonstrated that 14G3 exhibited a significant 10- to 20-fold increase in catalytic efficiency (kcat/Km) across the three substrates (benzyl alcohol, D-galactose and HMF), when compared to its parent. This enhancement is likely due to alterations in the active site architecture, as the N511S mutation is positioned in close proximity to this region. Nevertheless, the mutation introduced in 41C7 did not affect the catalytic performance, yet a 2-fold increase in protein production was observed. Both variants showed a slight loss in thermostability, suggesting a trade-off between activity and stability. Preliminary tests on immobilized enzymes in metal affinity resins showed that purified enzymes could be successfully attached to the carrier, however, a drop of activity was observed upon immobilisation. The nickel resin showed better results than cobalt resin, maintaining its activity even after 30 days of storage at 4 ºC. Further optimisation and development of enzyme immobilisation need to be performed to increase the efficiency of the enzyme when immobilised and, eventually, the stability to make it applicable for larger-scale applications in biotechnological industries and biorefineries. |
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